PROJECT SUMMARY Twenty percent of people in the United States will develop heart failure during their lives. Despite beneficial effects of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta blockers, and mineralocorticoid receptor antagonists on mortality, the five-year life expectancy of a patient with heart failure is fifty percent. In 2015, the FDA approved LCZ696 (Entresto?), a molecular complex of the ARB valsartan and sacubitril, a neprilysin (neutral endopeptidase-24.11) inhibitor prodrug, after LCZ696 reduced mortality compared to enalapril in a randomized clinical trial in patients with heart failure, reduced ejection fraction, and increased circulating brain natriuretic peptide (BNP) or N-terminal (NT) proBNP. LCZ696 also reduces rehospitalization in acutely decompensated heart failure. Nevertheless, LCZ696 has been underutilized in clinical practice, and concerns regarding hypotension have impeded use. The mechanism(s) through which the combined ARB and neprilysin inhibitor reduces blood pressure are not fully known. Neprilysin degrades many vasoactive peptides including the natriuretic peptides, angiotensins (Ang) I and II, endothelins, bradykinin, and substance P. In heart failure patients, LCZ696 increases BNP, a neprilysin substrate, while decreasing the non-neprilysin substrate NT-proBNP, suggesting that LCZ696 potentiates effects of the natriuretic peptide. On the other hand, natriuretic peptides are poor substrates for neprilysin compared to bradykinin and substance P, which can have beneficial effects on blood pressure, diuresis, natriuresis, fibrinolysis and remodeling but also contribute to adverse effects like hypotension and angioedema. Our research group has extensive experience studying the contribution of peptides to drugs that inhibit vasopeptidases such as ACE, dipeptidyl peptidase-4 (DPP4), and neprilysin. In this proposal, we test the overarching hypothesis that bradykinin contributes to vasodilator, blood pressure and renal effects of combined angiotensin receptor blockade/neprilysin inhibition. In Aim 1, we will test the hypothesis that LCZ696 potentiates the effects of intra-arterial bradykinin, substance P, or BNP compared to valsartan alone. We will test this hypothesis in the presence and absence of a DPP4 inhibitor, as it may enhance effects of neprilysin inhibition. In Aim 2, we will test the hypothesis that endogenous bradykinin contributes to hypotensive, natriuretic and diuretic effects of LCZ696 during initiation and up-titration in heart failure patients using a bradykinin B2 receptor antagonist. In Aim 3, we will probe the contribution of endogenous substance P to effects of LCZ696 using a substance P (NK1) receptor antagonist. In the combined Aims 2 and 3, we will assess individual patient factors such as race, gender, BNP (measured both by clinical immunoassay and by specific mass spectrometric assay), and NEP activity that predict blood pressure response to LCZ696. These studies will provide novel information about the mechanism(s) of action of combined angiotensin receptor blockade and neprilysin inhibition, and lead to new strategies to minimize adverse effects while maximizing beneficial effects of this promising new class of drugs for heart failure.